Patients with Familial Pulmonary Hypertension (FPAH) inherit heterozygous mutations in the BMP type 2 receptor BMPR2. It is unknown how these mutations cause pulmonary hypertension (PH). Half of these mutations escape Nonsense Mediated mRNA Decay and express BMPR2 mutant mRNA (NMD negative). Others are NMD positive and do not express mutant products. As NMD negative mutations express mutant gene products they may exert dominant inhibitory effects on the pulmonary vasculature. Recent evidence supports this: the age at diagnosis is younger in FPAH patients with NMD negative versus NMD positive BMPR2 mutations. This suggests NMD negative mutations cause more severe disease. Preliminary studies in mice carrying different Bmpr2 mutations suggest a mechanism by which this occurs. Mice with the heterozygous Bmpr2 Exon 2 deletion mutation (Bmpr2?Ex2/+) have more severe PH than mice carrying heterozygous null Bmpr2+/- mutations. This is associated with reduced eNOS activity and endothelial cell dysfunction in Bmpr2?Ex2/+ pulmonary vasculature. Over-expression of eNOS reverses the PH phenotype in Bmpr2?Ex2/+ mutant mice, indicating that eNOS deficiency plays a critical role in exacerbating PH. Furthermore, there is abnormal intracellular processing of the Bmpr2?Ex2 mutant product, and this interferes with eNOS function by abnormal intracellular sequestration of eNOS. As similar NMD negative Exon 2 deletion mutants are found in FPAH patients, these findings suggest that eNOS deficiency may be a common mechanism by which NMD negative BMPR2 mutations exacerbate PH in patients with FPAH. Three aims are proposed to test this hypothesis and define the mechanism by which this occurs. Aim 1 will compare vasoreactivity, PH responses and eNOS activity in the pulmonary vasculature of Bmpr2?Ex2/+ and Bmpr2+/- mice. Aims 2 will use cell culture techniques to evaluate how BMP signaling normally regulates eNOS expression and activity in pulmonary endothelial cells, while Aim 3 will compare eNOS regulation in Bmpr2+/- and Bmpr2?Ex2/+ mice, and will determine the molecular mechanism by which the Bmpr2?Ex2/+ mutant product exerts dominant inhibitory effects on eNOS activity in pulmonary endothelial cells. These studies will provide insight into how NMD positive FPAH BMPR2 mutations (like the Bmpr2+/- mutation) vs. NMD negative mutations (which are expressed and may behave like the Bmpr2?Ex2/+ mutation) modify vascular responses in patients with FPAH. By defining the mechanism by which Bmpr2?Ex2 inhibits eNOS they will also establish the foundation for therapeutic approaches to ameliorate PH by correcting NMD negative BMPR2 mutant processing defects in FPAH. PUBLIC HEALTH RELEVANCE: Pulmonary Arterial Hypertension is an invariably fatal disease for which there is no curative treatment. A major challenge for the scientific community therefore is to develop therapeutic approaches to prevent the progression of pulmonary vascular disease that develops in these patients. Our studies will provide important insight into a common pathway that regulates this process and identify approaches to interfere with this pathway and prevent the development of irreversible pulmonary vascular disease in these patients.